1. Field of the Invention
This invention relates to an isolated nucleic acid sequence having a nucleotide sequence that encodes a recombinant polypeptide having pancreatic lipase activity. This invention also relates to a recombinant vector and a recombinant host cell for producing the recombinant polypeptide. The recombinant polypeptide is adapted for preparation of an animal feed that is able to facilitate utilization of fats therein regarding pigs (especially postweaning piglets) and to enhance growth performance of the pigs.
2. Description of the Related Art
Generally, growth period of a pig can be divided into the following four phases: lactation phase, nursery phase, grower phase, and finisher phase. During the lactation phase, a newborn piglet (a body weight of about 1.5 kg) obtains energy from sow's milk. During the nursery phase, the weaned piglet (a body weight of about 6˜7 kg) grows and becomes a 5˜2-week-old nursery pig (a body weight of about 7˜30 kg). The nursery pig grows and becomes a 13˜20-week-old grower pig (a body weight of about 30˜60 kg) during the grower phase. The grower pig grows and becomes a 21˜30-week-old finisher pig (a body weight of about 60˜110 kg) during the finisher phase.
After entering the nursery phase, due to change of feed form, gastrointestinal tract development of the piglet is adversely influenced, e.g., insufficient secretion of digestive enzyme, reduction in absorption ability caused by change of intestinal villus structure, insufficient secretion of gastric acid, etc. Accordingly, feed intake and digestibility of the postweaning piglet are further decreased such that the postweaning piglet may undergo growth stasis (also known as postweaning lag).
In order to prevent energy deficiency phenomenon induced by postweaning lag, a method of maintaining caloric intake of postweaning piglets by adding various fat sources to a feed for the postweaning piglets is widely developed. A report has indicated that utilization of fats in a feed regarding postweaning piglets varies with the type of fats and the fat content. In contrast with animal fats, plant fats have a higher level of middle chain triglyceride (MCT) and unsaturated fatty acids such that the plant fats can be more easily digested and absorbed by postweaning piglets (K. R. Cera et al. (1988), J. Anim. Sci., 66:1430-1437; K. R. Cera et al. (1990), J. Anim. Sci., 68:2756-2765). However, in the second week after weaning, fat utilization of piglets is reduced on account of gradual decrease in pancreatic lipase activity, and the piglets are hence not capable of acquiring sufficient energy to maintain growth performance thereof (C. A. Flores et al. (1988), J. Pediatr. Gastroenterol. Nutr., 7:914-921; M. S. Jensen et al. (1997), J. Anim. Sci., 75:437-445). Accordingly, how to facilitate utilization of fats in a feed with respect to postweaning piglets is required to be investigated for the pig-farming industry and animal feed manufacturers so that the postweaning piglets are able to sufficiently obtain essential energy for growth.
Pancreatic lipase is an enzyme that is secreted by the pancreas, and is able to hydrolyze fat molecules. In the pancreas, newly formed pancreatic lipase is an inactive proenzyme that is composed of a signal peptide and a mature peptide, and that has 465 amino acid residues. Amino acid residues 1-16 constitute the signal peptide, and amino acid residues 17-465 constitute the mature peptide. When the signal peptide is subjected to cleavage processing by virtue of pancreatic protease, the inactive pancreatic lipase is converted to the active pancreatic lipase having fat-hydrolysis activity.
Due to rapid development of biotechnology, amino acid sequences of pancreatic lipases of numerous species and complete coding sequences thereof have been investigated and are available in the database at the NCBI (National Center for Biotechnology Information) website. For example, the following sequences can be found at the NCBI website: the amino acid sequence of pancreatic lipase of Homo sapiens (NCBI Accession No. 10835000) and the complete coding sequence thereof (NCBI Accession No. M93285); the amino acid sequence of pancreatic lipase of Mus musculus (NCBI Accession No. 37674236) and the complete coding sequence thereof (NCBI Accession No. BC061061); the amino acid sequence of pancreatic lipase of Rattus norvegicus (NCBI Accession No. 6981376) and the complete coding sequence thereof (NCBI Accession No. 6981375); the amino acid sequence of pancreatic lipase of Equus caballus (NCBI Accession No. 255653018) and the complete coding sequence thereof (NCBI Accession No. 255653017); and the amino acid sequence of pancreatic lipase of Canis familiaris (NCBI Accession No. 73998882) and the complete coding sequence thereof (NCBI Accession No. 50919159).
J. De Caro et al. isolated and purified porcine pancreatic lipase from porcine pancreatic tissues, subsequently conducted fragmentation on the porcine pancreatic lipase using CNBr to obtain peptide fragments, and acquired the amino acid sequence of the porcine pancreatic lipase by virtue of automated sequence analysis [J. De Caro et al. (1981), Biochim. Biophys. Acta, 671:129-138]. Even though the amino acid sequence of the porcine pancreatic lipase has been studied thoroughly, the applicants are unaware of any literature or prior art patent that has disclosed a complete coding sequence of the porcine pancreatic lipase.
In order to facilitate utilization of fats in a feed with respect to postweaning piglets, researchers in the related fields normally use pancreatic lipase directly obtained from the pancreas or various substances able to enhance pancreatic lipase activity as feed additives, and add the same into a feed for postweaning piglets so that activity of the pancreatic lipase in the postweaning piglets can be strengthened. For instance, U.S. Pat. No. 7,153,504 B2 has disclosed a stabilized pancreas product containing an emulsification of whole pancreas, which is blended with soy hulls. The emulsification of whole pancreas contains one or more pancreatic enzymes in zymogen form. The experimental results have indicated that the stabilized pancreas product can be used as an animal feed additive so as to facilitate utilization of nutrients with respect to an animal when the animal encounters a production change (e.g., a change in a feed or environment).
CN 101366451A discloses a feed additive for improving a fat digestion utilization ratio and a method for preparing the same. The feed additive contains the following components: 0-35 parts by weight of bile acid, 0-15 parts by weight of taurine, and 20-80 parts by weight of defatted rice bran. The experimental results reveal that: addition of a proper amount of bile acid and taurine into a feed is able to enhance activity of pancreatic lipase, to greatly improve the fat digestion utilization ratio, and to strengthen liver function, thereby lowering the incidence rate of animal fatty liver and the mortality of animals.
In an animal body, hydrolysis of fat molecules depends not only on pancreatic lipase, but also on pancreatic colipase. Therefore, adding pancreatic colipase to a feed for postweaning piglets is another possible way to enhance utilization of fats in the feed regarding the postweaning piglets. TW 200916584 discloses an isolated pancreatic colipase, and a method for enhancing growth performance of a pig using a feed additive containing the isolated pancreatic colipase. The experimental results indicate that: compared to the control group, during 4 weeks of feeding postweaning piglets with a feed containing the 5000 U/kg pancreatic colipase, the body weight of the postweaning piglets significantly increased at Days 15, 22, and 28; average daily gain (ADG) of the postweaning piglets significantly increased in the periods of Days 1-7 and Days 8-14, but is not significantly different from that of the control group in the periods of Days 15-21 and Days 22-28; and average daily feed intake (ADFI) and feed efficiency (FE) of the postweaning piglets are not significantly different from those of the control group during the overall period of the experiment.
Even though the aforementioned prior art patents can be applied to increase activity of pancreatic lipase in postweaning piglets so as to facilitate utilization of fats in a feed regarding the postweaning piglets (i.e., so as to facilitate conversion of the fats in the feed to essential needs for growth of the postweaning piglets), a long-term facilitation effect and a reduction in production cost of the feed are still in demand. Furthermore, most of conventional methods for investigating porcine pancreatic lipase employ a protein purification step to purify the porcine pancreatic lipase from frozen pancreatic tissues. However, the protein purification step is time-consuming and complicated, and the amount of the thus obtained porcine pancreatic lipase is insufficient. Therefore, using the porcine pancreatic lipase obtained by the aforesaid conventional methods as a feed additive for postweaning piglets may give rise to a high production cost. In addition, a production cost of the artificial porcine pancreatic lipase produced using the conventional peptide synthesis technology may be higher than that of the porcine pancreatic lipase obtained by the aforesaid protein purification step.
In order to overcome the drawbacks of the prior art patents, genetic engineering technology may be used to clone the coding gene of porcine pancreatic lipase, thereby inducing mass production of recombinant porcine pancreatic lipase. Accordingly, the applicants attempted to clone porcine pancreatic lipase gene (pLip gene) from porcine pancreatic tissues, and to employ genetic engineering technology for rapidly and massively producing recombinant porcine pancreatic lipase. The applicants used the obtained recombinant porcine pancreatic lipase to prepare an animal feed and fed the postweaning piglets with the animal feed. The applicants subsequently found that: the postweaning piglets were able to effectively utilize fats in the animal feed as an energy source, and growth performance of the postweaning piglets was enhanced accordingly.